Harvey, Kirsten and N ras proteins (termed H-ras, K-ras, and N-ras, respectively) are immunologically related proteins and are collectively termed p21. They are products of the ras family of cellular genes which are found in a wide variety of nucleated mammalian cells. The ras genes appear to be frequent targets of genetic alterations that can lead normal cells along the pathway to malignancy. Ras oncogenes have been identified in a wide array of premalignant and malignant cells.
The p21 proteins consist of about 188-189 amino acids having a molecular weight of about 21,000 daltons. Viral and cellular ras genes encode membrane bound proteins (Willingham et al., Cell 19;1005 (1980)) which bind guanine nucleotides (Schlonick et al., PNAS (USA) 76:5355 (1979); Papageorge et al., J. Virol. 44:509 (1982); and Fine et al., Cell 37:151(1984)) and possess intrinsic GTPase activity (McGrath et al., Nature 301:644 (1984); Sweet et al., Nature 311:273 (1984); Gibbs et al., PNAS (USA) 81:5704 (1984); and Manne et al., PNAS 82:376 (1985)).
DNA mediated transfection experiments using NIH3T3 cells as recipients have led to the identification of a family of activated transforming genes homologous to the ras genes of the H-ras and K-ras sarcoma viruses. A third member of the ras family designated N-ras has been identified but has not been found to have a retroviral counterpart. Activated (mutated) ras genes are structurally distinct from their normal homologs, having amino acid substitutions in the protein at positions 12, 13, or 61. (Tabin et al., Nature 300:143 (1982); Reddy et al., Nature 300:149 (1982); Bos et al., Nature 315:716 (1985); Yuasa et al., Nature, 303:775-779 (1983); Der et al., cell 44:167-176 (Jan. 17, 1986)). Taparowsky et al., Banbury Report, 14:123-133 (1983) cited in Chem. Abstracts CA 100(1):1425n, teaches that the change at residue 12 from N-terminus of the H ras p21 from glycine to valine is sufficient to convert the normal protein to a transforming protein.
Shimizu et al., Nature 304:497-500 (1983) cited in Chem. Abstracts 99(19):1530936, teaches the presence of a cysteine residue at amino acid 12 in the human lung cancer cell line calu-1 homolog of the v-Ki-ras gene. Fasano et al., J. Mol. Appl. Genet., 2(2):173-180, cited in Chem. Abstracts CA 99(19):153080v, teaches that the T24 H-ras-1 gene product is nearly identical to the v-H-ras p21 transforming protein encoded by Harvey sarcoma virus. Recent reports have shown the presence of activated ras p21 proteins in 40-50% of human colorectal cancers and preneoplastic lesions of the colon termed adenomas (Bos et al., Nature 327:293 (1987), Forrester et al., Nature 327:299 (1987) and Volgelstein et al., NEJM 319:525 (September 1988)). Recent studies have also shown expression of activated ras genes and mutated ras p21 proteins in 20-30% of lung carcinomas (Rodenhuis et al., Cancer Res., 48:5738 (1988)) and over 90% of pancreatic carcinomas (Almoguera et al., Cell 53:549 (1988)). In certain forms of leukemia, such as acute myelogeneous leukemia and in certain preleukemic states, activated ras p21 proteins have been described.
These activated ras genes and mutated proteins have also been found in established cell lines as well as primary and metastatic tumors. Gambke et al., Nature 307:476, (1984), demonstrated a transforming N-ras gene in bone marrow cells from a patient aith acute myeloblastic leukemia ("AML"). In contrast, DNA from fibroblast cells from the same patient was not transforming.
The p21 ras protein in its normal nonactivated form contains the glycine amino acid at positions 12 and 13 and the glutamine amino acid at position 61. The p21 protein found in normal cells has the following primary amino acid structure for the amino acid sequence 5 to 19: .sup.5 Lysine-leucine-valine-valine-valine-glycine-alanine-glycine-glycine-valine -glycine-lysine-serine-alanine-leucine.sup.19.
Ras proteins act as molecular switches relaying proliferative signals from cell surface receptors to the nucleus and cytoskeleton. Activation of these receptors leads to the activation of a guanines nucleotide exchange factor, which induces the exchange of guanine diphosphate ("GDP") for guanine triphosphate ("GTP"). Specifically, the activation of Ras by the binding of GTP is required for the ability of many growth factors and cytokines to induce non-proliferating cells to enter G1 phase of the cell cycle. Activation of membrane-bound Ras by growth factor and cytokine receptors is generally achieved by the recruitment of Grb2-Sos complexes to the receptors themselves or to adaptor proteins such as Shc.
A primary target of activated Ras during growth factor stimulation is Raf, which is the first component of a protein kinase cascade that leads to activation of the MAP kinases Erk1 and Erk2 (Avruch et al., "Raf Meets Ras: Completing the Framework of a Signal Transduction Pathway," Trends Biochem. Sci., 19:279-83 (1994)). The phosphorylation of transcription factors by these MAP kinases results in the expression of immediate early response genes, such as c-fos, that are required for early G1 progression. Although these signalling events occur within minutes of growth factor stimulation, microinjection of neutralizing anti-Ras antibodies in late G1 phase blocks progression of fibroblasts into S phase (Mulcahy, et al., "Requirement for Ras Proto-oncogene Function During Serum-Stimulated Growth of NIH 3T3 Cells, Nature, 313:214-43 (1985)). Furthermore, studies using combinations of cell cycle inhibitors and anti-Ras microinjection clearly demonstrate multiple points of Ras requirement in early and late G1 phase (Dobrowolski et al., "Cellular Ras Activity Is Required for Passage Through Multiple Points of the G-0-G-1 Phase in BALB-c 3T3 Cells," Molecular and Cellular Biology, 14:5441-49 (1994). These findings, together with the observations that expression of oncogenic Ras increases cyclin D1 levels and shortens G1 phase (Liu, et al., "Ras Transformation Results in an Elevated Level of Cyclin D1 and Acceleration of G1 Progression in NIH 3T3 Cells," Mol. Cell Biol., 15:3654-63 (1995); Winston et al., "Regulation of the Cell Cycle Machinery by Oncogenic Ras," Oncogene, 12:127-34 (1996)) and that Ras and cyclin D1 cooperate in cellular transformation assays (Hinds et al., "Function of a Human Cyclin Gene as an Oncogene," Proc. Natl. Sci. USA, 91:709-13 (1994); Lovec et al., "Oncogenic Activity Cyclin D1 Revealed Through Cooperation with Ha-ras; Link Between Cell Cycle Control and Malignant Transformation," Oncocene, 9:323-26 (1994)) point to an important role for Ras in regulating progression from G1 into S phase.
However, important questions remain as to whether Ras controls signalling everts during cell cycle progression, and, if so, at which point in the cell cycle it is activated. The Ras proteins function by cycling between active and inactive forms; in the active form Ras binds to GTP and is converted to the inactive form by conversion of GTP to GDP. Activation of Ras is promoted by numerous extracellular signals such as growth factors, and, when activated, Ras specifically interacts with intracellular targets to transduce growth stimulatory signals from the cell's exterior to the nucleus. One such target of activated Ras in the Raf-1 proto-oncoprotein, a protein kinase involved in signalling to the nucleus.
The mutations of ras gene., that occur in human cancer, as discussed above, cause a constitutive activation of the Ras protein, and the resulting deregulation of growth control is believed to contribute to the cancer process. Furthermore, it is known that ras gene mutation occurs at a particular: stage in the multi-step process of colon cancer progression, and it is likely that ras mutations might occur at defined, perhaps early, stages in other types of cancer. Accordingly, the detection of ras activation in human tumors might be of great diagnostic and prognostic use.
Most previous analyses of Ras activation have measured the GTP:GDP ratio of immunoprecipitated Ras following [32P] radiolabelling of cells (Gibbs et al., "Modulation Of Guanine Nucleotides Bound to Ras in Nih3t3 Cells by Oncogenes Growth Factors and the Gtpase Activating Protein Gap," J. Biol. Chem., 265:20 437-42 (1990); Satoh, et al., "Platelet-Derived Growth Factor Stimulates Formation of Active p21ras-GTP Complex in Swiss Mouse 3T3 Cells," Proc. Natl. Acad. Sci. USA, 87:59 93-97 (1990); Gibbs, J. B., "Determination of Guanine Nucleotides Bound to Ras in Mammalian Cells," Methods Enzymol., 255:118-25 (1995); Satoh, et al., "Measurement of Ras-Bound Guanine Nucleotides in Stimulated Hematopoietic Cells," Methods Enzymol., 255:149-55 (1995). The ability of even very low levels of radioisotopes to cause rapid (i.e., within a few hours) cell cycle arrest or apoptosis (Wimber D. E., "Effects of Intracellular Irradiation with Tritium," Adv. Radiat. Biol., 1:85-115 (1964); Dover et al., "p53 Expression in Cultured Cells Following Radioisotope Labelling," J. Cell Sci., 107:1181-84 (1994); Yeargin, et al., "Elevated Levels of Wild-Type p53 Induced by Radiolabelling of Cells Leads to Apoptosis or Sustained Growth Arrest," Current Biology, 5:423-31 (1995), however, precludes the use of such assays to measure Ras activity in cycling cells.
Previous assays of activated Ras, such as U.S. Pat. No. 5,443,956 to Carney, have employed antibodies specific for particular activated Ras mutants. This type of assay suffers the drawbacks of not detecting all potential activating mutations and rot detecting Ras activation in response to activation of other oncogenes.
Chuang et al., "Critical Binding and Regulating Interactions Between Ras and Raf Occur Through a Small, Stable N-Terminal Domain of Raf and Specific Ras Effector Residues," Molecular & Cellular Biology, 14(8):5318-325 (1994); Warne et al., "Direct Interaction of Ras and the Amino-terminal Region of Raf-1 in vitro, Nature, 364:352-355 (1993); and Ghosh et al., "The Cysteine-rich Region of Raf-1 Kinase Contains Zinc, Translocates to Liposomes, and Is Adjacent to a Segment That Binds GTP-Ras," J. Biological Chem., 269(13):10000-10007 (1994) study the interaction of the GTP-bound activated ras protein to an raf-1-GST fusion protein using purified recombinantly produced GTP-ras. Thus, these references did not study reactions using lysates from cell cultures, such as cells taken from cancerous tissue. One would have expected that the many proteins and other components present in a lysate would interfere with the binding of GTP-ras to raf-1. Furthermore these references studied Ras-Raf interaction under optimized conditions, for instance maximized GTP "loading" of Ras and, therefore, were unable to study the effects of complex cellular regulatory networks on Ras activation. These references also employed recombinant sources of Ras which, therefore, were not subject to the post-translational modifications of Ras that occur in mammalian cells.
The present invention is directed to overcoming these deficiencies.